Lipid-dependent Generation of Dual Topology for a Membrane Protein

The mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is unknown. We posit that such duality is determined by both the protein sequence and the membrane lipid composition wherein a spatial or temporal change in the latter can result in a post-assembly change in protein structure and function. To investigate whether co-existence of multiple topological conformers is dependent on the membrane lipid composition, we determined the topological organization of lactose permease in an Escherichia coli model cell system in which phosphatidylethanolamine membrane content can be systematically varied. At intermediate levels of phosphatidylethanolamine a mixture of native and topologically mis-oriented conformers co-existed. There was no threshold level of phosphatidylethanolamine determining a sharp transition from one conformer to the other. Co-existing conformers were not in rapid equilibrium at a static lipid composition indicating that duality of topology is established during an early folding step. Depletion of intermediate levels of phosphatidylethanolamine after final protein assembly resulted in complete mis-orientation of the native conformer. Combined with previous results, such topological dynamics are reversible in both directions. We propose a thermodynamically based model for how lipid-protein interactions can result in a mixed topological organization and how changes in lipid composition can result in changes in the ratio of topologically distinct conformers of proteins. These observations demonstrate a potential lipid-dependent biological switch for generating dynamic structural and functional heterogeneity for a protein within the same membrane or between different membranes in more complex eukaryotic cells.     

NMR studies of metal ion binding to the Zn-finger-like HNH motif of colicin E9

The 134 amino acid DNase domain of colicin E9 contains a zinc-finger-like HNH motif that binds divalent transition metal ions. We have used 1D 1H and 2D 1H-15N NMR methods to characterise the binding of Co2+, Ni2+ and Zn2+ to this protein. Data for the Co2+-substituted and Ni2+-substituted proteins show that the metal ion is coordinated by three histidine residues; and the NMR characteristics of the Ni2+-substituted protein show that two of the histidines are coordinated through their N(epsilon2) atoms and one via its N(delta1). Furthermore, the NMR spectrum of the Ni2+-substituted protein is perturbed by the presence of phosphate, consistent with an X-ray structure showing that phosphate is coordinated to bound Ni2+, and by a change in pH, consistent with an ionisable group at the metal centre with a pKa of 7.9. Binding of an inhibitor protein to the DNase does not perturb the resonances of the metal site, suggesting there is no substantial conformation change of the DNase HNH motif on inhibitor binding. 1H-15N NMR data for the Zn2+-substituted DNase show that this protein, like the metal-free DNase, exists as two conformers with different 1H-15N correlation NMR spectra, and that the binding of Zn2+ does not significantly perturb the spectra, and hence structures, of these conformers beyond the HNH motif region.     

Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy

We applied hydrostatic pressure perturbation to study substrate-induced transitions in human cytochrome P450 3A4 (CYP3A4) with bromocriptine (BCT) as a substrate. The barotropic behavior of the purified enzyme in solution was compared with that observed in recombinant microsomes of Saccharomyces cerevisiae coexpressing CYP3A4, cytochrome b(5), (b(5)) and NADPH-cytochrome P450 reductase (CPR). Important barotropic heterogeneity of CYP3A4 was detected in both cases. Only about 70% of CYP3A4 in solution and about 50% of the microsomal enzyme were susceptible to a pressure-induced P450-->P420 transition. The results suggest that both in solution and in the membrane CYP3A4 is represented by two conformers with different positions of spin equilibrium and different barotropic properties. No interconversion between these conformers was observed within the time frame of the experiment. Importantly, a pressure-induced spin shift, which is characteristic of all cytochromes P450 studied to date, was detected in CYP3A4 in solution only; the P450-->P420 transition was the sole pressure-induced process detected in microsomes. This fact suggests unusual stabilization of the high-spin state of CYP3A4, which is assumed to reflect decreased water accessibility of the heme moiety due to specific interactions of the hemoprotein with the protein partners (b(5) and CPR) and/or membrane lipids.     

Slow-folding kinetics of ribonuclease-A by volume change and circular dichroism: evidence for two independent reactions.

The slow refolding of guanidine-HCl-denatured ribonuclease-A was studied by volume change and by kinetic CD at 222 and 276 nm. Dilatometric measurements revealed that on refolding there is a fast volume change of +232 mL/mol of protein. This is followed by a very slow nonexponential change that takes about 25 min to reach equilibrium. By adding varying amounts of (NH4)2SO4, the slow volume change curve was resolved into 2 concurrent reactions. The faster of the 2 slow events entails a negative volume change of -64 mL/mol of protein and appears to arise from proline isomerization. The slower process, attended by a positive change of +53 mL/mol of protein, has properties consistent with the "XY" reaction of Lin and Brands (1983, Biochemistry 22:563-573). This reaction is so named because the conformational nature of neither its initial (Y) nor its final state (X) is known; the transition is characterized solely by its absorbance and fluorescence kinetics. These are the first direct physical measures attributable to the "XY" process. The early formation of a compact structure in the event responsible for the rapid +232-mL/mol volume change, however, is consistent with the sequential model of folding (Cook KH, Schmid FX, Baldwin RL, 1979, Proc Natl Acad Sci USA 76:6157-6161; Kim PS, Baldwin RL, 1980, Biochemistry 19:6124-6129). The usefulness of volume change measurements as a method of detecting structural rearrangements was confirmed by finding agreement between time constants obtained from parallel volume change and kinetic CD experiments. The measured volume changes arise from both changes in hydration and changes in the packing of atoms in the interior of the protein.     

Protein conformer selection by ligand binding observed with crystallography.

A large-scale movement between "closed" and "open" conformations of a protein loop was observed directly with protein crystallography by trapping individual conformers through binding of an exogenous ligand and characterization with solution kinetics. The buried indole ring of Trp191 in cytochrome c peroxidase (CCP) was displaced by exogenous ligands, causing a conformational change of loop Pro190-Asn195 and exposing Trp191 to the protein surface. Kinetic measurements are consistent with a two-step binding mechanism in which the rate-limiting step is a transition of the protein to the open state, which then binds the ligand. This large-scale conformational change of a functionally important region of CCP is independent of ligand and indicates that about 4% of the wild-type protein is in the open form in solution at any given time.     

Structural aspects of Ca2+ binding by acyclic peptides: low-energy conformational domains and molecular dynamics of N-acetyl-L-prolyl-D-alanyl-L-alanine-N'-methylamide.

We have applied random-search, energy minimization and molecular dynamics simulations to investigate the structural aspects of the interaction of N-acetyl-L-prolyl-D-alanyl-L-alanine-N'-methylamide with Ca2+. Spectral data on related peptides had suggested that the beta-turn conformation might be a prerequisite for the binding of cation ion by such short linear peptides. In order to relate the conformational characteristics with the Ca(2+)-binding affinities of these peptides, the molecular events involved in cation binding need to be understood. We have addressed this problem in this study by using a systematic approach that involved the following steps. First, a random search technique was used to generate a large population of conformers for the free peptide in the absence of Ca2+. Next, the energies of these conformers were computed. Conformations with energies within 4 kcal/mol of the global minimum were analysed and found to fall into four main groups characterized by the presence of different types of hydrogen-bonded structures including single and consecutive beta-turns. The energies for interconversion of conformers from one group to another were computed and found to be relatively small (< 10 kcal/mol). Finally, molecular dynamics of the peptide at 300K in the presence of Ca2+ were used to simulate the cation binding process. Starting points for these simulations were generated by placing the ion in the vicinity of two molecules of the peptide. The simulation results showed that the conformers with two consecutive beta-turns led to the formation of a stable 2:1 (peptide:Ca2+) sandwich complex in agreement with earlier experimental observations on similar linear peptides. While the starting conformation of the peptide in the consecutive beta-turn structure allowed for the proper orientation of three carbonyl oxygen atoms for chelation to the metal ion, the dynamics of complex formation rearranged the peptide structure substantially, leading to the formation of an 8-coordinated Ca2+ complex in a dodecahedral spatial arrangement. Thus, based on the energetics of the structures and processes involved, the present study demonstrates that: a) peptide-Ca2+ complex formation is initiated by conformers adopting consecutive beta-turn structures which subsequently go over to a significantly different conformation found in the complex; and, b) The facile interconversion between the low-energy conformers in the different groups would help shift the equilibrium population towards the consecutive beta-turn structure during the complex formation.     

Circular dichroism eigenspectra of polyproline II and β-strand conformers of trialanine in water: Singular value decomposition analysis

Despite that a number of experimental and theoretical investigations have been carried out to determine the structure of trialanine in water, the reported populations of polyproline II (PPII) and β-strand conformers vary and were found to be dependent on which spectroscopic method was used. Such discrepancies are due to limitations of different spectroscopic methods used. Here, the temperature- and pH-dependent circular dichroism (CD) and NMR experiments have been carried out to develop a self-consistent singular value decomposition procedure. The temperature-dependent CD spectra indicate the presence of two conformers, but due to the two peptide bonds in a trialanine, one should take into consideration of four different conformers to fully interpret the NMR results. From the pH-dependent NMR coupling constant measurements, the conformation of zwitterionic trialanine is little different from that of cationic one. The strong pH dependency of CD spectrum is likely due to charge transfer transitions between carboxylate and nearby peptide groups or internal field effects not to pH-dependent conformational change. To simultaneously analyze the temperature-dependent CD and NMR data, a self-consistent procedure was used to newly determine the reference NMR coupling constants required to estimate one of the peptide dihedral angles. From the estimated enthalpy and entropy changes associated with the transition from enthalpically favorable PPII conformer to entropically favorable β-strand conformer, the relative populations of the four possible conformers of trialanine were determined and compared with the previous experimental findings. We anticipate that the present experimental results and interpretation procedure would be of use in determining the solution structures of small oligopeptides in the future.     

The calorimetric criterion for a two-state process revisited.

The "calorimetric criterion" is one of the important experimental approaches for determining whether protein folding is an "all-or-none" two-state transition (i.e., whether intermediates are present at equilibrium). The calorimetric criterion states that the equivalence of the "measured" calorimetric enthalpy change and the effective two-state van't Hoff enthalpy change demonstrates that there is a two-state transition. This paper addresses the essential question of whether the calorimetric criterion is a necessary and sufficient condition for a two-state process and shows that it is necessary but not sufficient by means of specific examples. Analysis of simple models indicates that the heat capacity curve, regardless of whether it originates from a two-state process or not, can always be decomposed in such a way that the calorimetric criterion is satisfied. Exact results for a three-state model and a homopolymer tetramer demonstrate that the deviation from the calorimetric criterion is not simply related to the population of intermediate states. Analysis of a three-helix bundle protein model, which has a two-state folding from a random coil to ordered (molten) globule, shows that the calorimetric criterion may not be satisfied if the standard linear interpolation of baselines (weighted or unweighted) is employed. A specific example also suggests that the more recently introduced deconvolution method is not necessarily better than the simple calorimetric criterion for distinguishing a two-state transition from a three-state transition. Although the calorimetric criterion is not a sufficient condition for a two-state process, it is likely to continue to be of practical utility, particularly when its results are shown to be consistent with those from other experimental methods.     

Finding a needle in the haystack: computational modeling of Mg2+ binding in the active site of protein farnesyltransferase

Studies aimed at elucidating the unknown Mg2+ binding site in protein farnesyltransferase (FTase) are reported. FTase catalyzes the transfer of a farnesyl group to a conserved cysteine residue (Cys1p) on a target protein, an important step for proteins in the signal transduction pathways (e.g., Ras). Mg2+ ions accelerate the protein farnesylation reaction by up to 700-fold. The exact function of Mg2+ in catalysis and the structural characteristics of its binding remain unresolved to date. Molecular dynamics (MD) simulations addressing the role of magnesium ions in FTase are presented, and relevant octahedral binding motifs for Mg2+ in wild-type (WT) FTase and the Dβ352A mutant are explored. Our simulations suggest that the addition of Mg2+ ions causes a conformational change to occur in the FTase active site, breaking interactions known to keep FPP in its inactive conformation. Two relevant Mg2+ ion binding motifs were determined in WT FTase. In the first binding motif, WT1, the Mg2+ ion is coordinated to D352β, zinc-bound D297β, two water molecules, and one oxygen atom from the α- and β-phosphates of farnesyl diphosphate (FPP). The second binding motif, WT2, is identical with the exception of the zinc-bound D297β being replaced by a water molecule in the Mg2+ coordination complex. In the Dβ352A mutant Mg2+ binding motif, D297β, three water molecules, and one oxygen atom from the α- and β-phosphates of FPP complete the octahedral coordination sphere of Mg2+. Simulations of WT FTase, in which Mg2+ was replaced by water in the active site, recreated the salt bridges and hydrogen-bonding patterns around FPP, validating these simulations. In all Mg2+ binding motifs, a key hydrogen bond was identified between a magnesium-bound water and Cys1p, bridging the two metallic binding sites and, thereby, reducing the equilibrium distance between the reacting atoms of FPP Cys1p. The free energy profiles calculated for these systems provide a qualitative understanding of experimental results. They demonstrate that the two reactive atoms approach each other more readily in the presence of Mg2+ in WT FTase and mutant. The flexible WT2 model was found to possess the lowest barrier toward the conformational change, suggesting it is the preferred Mg2+ binding motif in WT FTase. In the mutant, the absence of D352β makes the transition toward a conformational change harder. Our calculations find support for the proposal that D352β performs a critical role in Mg2+ binding and Mg2+ plays an important role in the conformational transition step.     

Defining the physical gate of a mechanosensitive channel, MscL, by engineering metal-binding sites

The mechanosensitive channel of large conductance, MscL, of Escherichia coli is one of the best-studied mechanosensitive proteins. Although the structure of the closed or "nearly-closed" state of the Mycobacterium tuberculosis ortholog has been solved and mechanisms of gating have been proposed, the transition from the closed to the open states remains controversial. Here, we probe the relative position of specific residues predicted to line the pore of MscL in either the closed state or during the closed-to-open transition by engineering single-site histidine substitutions and assessing the ability of Ni2+, Cd2+ or Zn2+ ions to affect channel activity. All residues predicted to be within the pore led to a change in channel threshold pressure, although the direction and extent of this change were dependent upon the mutation and metal used. One of the MscL mutants, L19H, exhibited gating that was inhibited by Cd2+ but stimulated by Ni2+, suggesting that these metals bind to and influence different states of the channel. Together, the results derived from this study support the hypotheses that the crystal structure depicts a "nearly closed" rather than a "fully closed" state of MscL, and that a clockwise rotation of transmembrane domain 1 occurs early in the gating process.     

Hydration structure of antithrombin conformers and water transfer during reactive loop insertion.

The serine protease inhibitor antithrombin undergoes extensive conformational changes during functional interaction with its target proteases. Changes include insertion of the reactive loop region into a beta-sheet structure in the protein core. We explore the possibility that these changes are linked to water transfer. Volumes of water transferred during inhibition of coagulation factor Xa are compared to water-permeable volumes in the x-ray structure of two different antithrombin conformers. In one conformer, the reactive loop is largely exposed to solvent, and in the other, the loop is inserted. Hydration fingerprints of antithrombin (that is, water-permeable pockets) are analyzed to determine their location, volume, and size of access pores, using alpha shape-based methods from computational geometry. Water transfer during reactions is calculated from changes in rate with osmotic pressure. Hydration fingerprints prove markedly different in the two conformers. There is an excess of 61-76 water molecules in loop-exposed as compared to loop-inserted conformers. Quantitatively, rate increases with osmotic pressure are consistent with the transfer of 73 +/- 7 water molecules. This study demonstrates that conformational changes of antithrombin, including loop insertion, are linked to water transfer from antithrombin to bulk solution. It also illustrates the combined use of osmotic stress and analytical geometry as a new and effective tool for structure/function studies.     

Theoretical study of the complementarity in halogen–bonded complexes involving nitrogen and halogen as negative sites

The conformations of trialkylphosphates (alkyl = propyl, butyl, pentyl and hexyl) in various diluents were studied by molecular dynamics simulations. The population density of various conformers of trialkylphosphate in different diluents such as water and n-dodecane was determined. The Helmholtz energy change accompanied by the transition between various conformations was computed. The aggregation behavior of tributylphosphate in water and water-dodecane medium was studied.     

Thermodynamics of the alkaline transition of cytochrome c.

The apparent equilibrium constant (Kapp) of the alkaline transition (AT) of beef heart cytochrome c, obtained from pH titrations of the current intensities in cyclic voltammetry experiments, has been measured as a function of the temperature from 5 to 65 degrees C, at different ionic strength (I = 0.01-0.2 M). The temperature profile of the pKapp values is biphasic and yields two distinct sets of DeltaH degrees 'AT and DeltaS degrees 'AT values below and above approximately 40 degrees C. In the low-temperature range, the process is endothermic and is accompanied by a small positive entropy change, while at higher temperatures it becomes less endothermic and involves a pronounced entropy loss. The temperature dependence of the transition thermodynamics is most likely the result of the thermal transition of native ferricytochrome c from a low-T to an high-T conformer which occurs at alkaline pH values at a temperature comparable with above (Ikeshoji, T., Taniguchi, I., and Hawkridge, F. M. (1989) J. Electroanal. Chem. 270, 297-308; Battistuzzi, G., Borsari, M., Sola, M., and Francia, F. (1997) Biochemistry 36, 16247-16258). Thus, it is apparent that the transitions of the two native conformers to the corresponding alkaline form(s) are thermodynamically distinct processes. It is suggested that this difference arises from either peculiar transition-induced changes in the hydration sphere of the protein or to the preferential binding of different lysines to the heme iron in the two temperature ranges. Extrapolation of the Kapp values at null ionic strength allowed the determination of the thermodynamic equilibrium constants (Ka) at each temperature, hence of the "true" standard thermodynamic parameters of the transition. The pKa value at 25 degrees C was found to be 8.0. A pKapp value of 14.4 was calculated for the alkaline transition of ferrocytochrome c at 25 degrees C and I = 0.1 M. The much greater relative stabilization of the native state in the reduced as compared to the oxidized form turns out to be almost entirely enthalpic in origin, and is most likely due to the greater affinity of the methionine sulfur for the Fe(II) ion. Finally, it is found that the Debye-Hückel theory fits the ionic strength dependence of the pKapp values, at least qualitatively, as observed previously for the ionic strength dependence of the reduction potential of this protein class. It is apparent that the increase in the pKapp values with increasing ionic strength is for the most part an entropic effect.     

A toxic monomeric conformer of the polyglutamine protein

Polyglutamine (polyQ) diseases are classified as conformational neurodegenerative diseases, like Alzheimer and Parkinson diseases, and they are caused by proteins with an abnormally expanded polyQ stretch. However, conformational changes of the expanded polyQ protein and the toxic conformers formed during aggregation have remained poorly understood despite their important role in pathogenesis. Here we show that a beta-sheet conformational transition of the expanded polyQ protein monomer precedes its assembly into beta-sheet-rich amyloid-like fibrils. Microinjection of the various polyQ protein conformers into cultured cells revealed that the soluble beta-sheet monomer causes cytotoxicity. The polyQ-binding peptide QBP1 prevents the toxic beta-sheet conformational transition of the expanded polyQ protein monomer. We conclude that the toxic conformational transition, and not simply the aggregation process itself, is a therapeutic target for polyQ diseases and possibly for conformational diseases in general.     

Highly fluctuating protein structures revealed by variable-pressure nuclear magnetic resonance

Although our knowledge of basic folded structures of proteins has dramatically improved, the extent of our corresponding knowledge of higher-energy conformers remains extremely slim. The latter information is crucial for advancing our understanding of mechanisms of protein function, folding, and conformational diseases. Direct spectroscopic detection and analysis of structures of higher-energy conformers are limited, particularly under physiological conditions, either because their equilibrium populations are small or because they exist only transiently in the folding process. A new experimental strategy using pressure perturbation in conjunction with multidimensional NMR spectroscopy is being used to overcome this difficulty. A number of rare conformers are detected under pressure for a variety of proteins such as the Ras-binding domain of RalGDS, beta-lactoglobulin, dihydrofolate reductase, ubiquitin, apomyoglobin, p13(MTCP1), and prion, which disclose a rich world of protein structure between basically folded and globally unfolded states. Specific structures suggest that these conformers are designed for function and are closely identical to kinetic intermediates. Detailed structural determination of higher-energy conformers with variable-pressure NMR will extend our knowledge of protein structure and conformational fluctuation over most of the biologically relevant conformational space.     

FTIR spectroscopic study of the dynamics of conformational substates in hydrated carbonyl-myoglobin films via temperature dependence of the CO stretching band parameters.

Two hydrated carbonyl myoglobin (MbCO) films, one containing (0.30 g water)/(g MbCO) from MbCO solution in water at pH 5.5 and the other (0.32 g water)/(gMbCO) from 0.1 M potassium phosphate buffer solution at pH 6.8, were studied by FTIR spectroscopy from 293 K to 78 K at selected temperatures on cooling and reheating. Above approximately 180 K the general trend in temperature dependence of half-bandwidths, peak maxima, and band area ratios of the A1 and A3 conformer bands is similar to those reported by Ansari et al. (1987. Biophys. J. 26:337) for MbCO in 75% glycerol/water solution, but abrupt changes in slopes at approximately 180-200 K and freezing-in of conformer populations, which could be taken as indicator for glass transition of the solvent or the protein, are absent for the hydrated MbCO films. This is interpreted in terms of an exceptionally broad distribution of relaxation times, and is in accord with conclusions from recent calorimetric annealing studies of hydrated protein powders (Sartor et al. 1994. Biophys. J. 66:249). Exchange between the three A conformers does not stop at approximately 180-200 K but occurs over the whole temperature region studied. These results are then discussed with respect to MbCO's behavior in the glass-->liquid transition region of glass-forming solvents, and it is concluded that, in analogy to the behavior of low-molecular-weight compounds with a distribution of rapidly interconverting conformers, freezing-in of MbCO's A conformer populations by the solvent should not be mistaken for a glass transition of MbCO.     

The relationship between the cation and protein content, cationic transport and the rate of protein synthesis detected in cells in the presence and in the absence of ouabain]

The measurements were made of monovalent cation (K+ + Rb+, Na+) and protein contents, of leucin incorporation into the protein (protein synthesis), and of ouabain-sensitive K+ influx during the growth of the Jurkat culture cells in the presence or in the absence of ouabain. All the data were calculated per cell. The time dependences of these parameters are nonlinear [correction of unline] curves. The results of three independent measurements are not reproduced. The problem was to find out an intracellular "main" parameter, so that the dependences between this and the rest of parameters be of line character. It has been found that it is the protein synthesis rate that may be taken as the main parameter, because between the protein synthesis rate and all the other parameters there is the line connection. Comparative results obtained with or without ouabain showed that K+ and Na+ concentrations in the cell were different, although the constants of the rate of protein synthesis were the same. This points to the same type of change of the protein synthesis process in the cells growing with or without ouabain.     

Phenylisoserine in the gas-phase and water: Ab initio studies on neutral and zwitterion conformers

The conformational landscape of phenylisoserine (PhIS) was studied. Trial structures were generated by allowing for all combinations of single-bond rotamers. Based on the B3LYP/aug-cc-pVDZ calculations 54 conformers were found to be stable in the gas phase. The six most stable conformers were further optimized at the B3LYP/aug-cc-pVTZ and MP2/aug-cc-pVDZ levels for which characteristic intramolecular hydrogen bond types were classified. To estimate the influence of water on PhIS conformation, the IEF-PCM/B3LYP/aug-cc-pVDZ calculations were carried out and showed 51 neutral and six zwitterionic conformers to be stable in water solution. According to DFT calculations, the conformer equilibrium in the gas phase is dominated by one conformer, whereas the MP2 calculations suggest three PhIS structures to be significantly populated. Comparison of DFT and MP2 energies of all 57 structures stable in water indicates that, in practice, one zwitterionic and one neutral conformer determine the equilibrium in water. Based on the AIM calculations, we found that for the neutral conformers in vacuum and in water, d(H...B) is linearly correlated with Laplacian at the H-bond critical point.     

Conformational equilibria in monomeric alpha-synuclein at the single-molecule level

Human alpha-Synuclein (alphaSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of alphaSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between alphaSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy-based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant alphaSyn. The conformational heterogeneity of monomeric alphaSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and "beta-like" structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the "beta-like" structures significantly increased in different conditions promoting the aggregation of alphaSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric alphaSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric alphaSyn.     

Role of protein conformation changes and transphosphorylations in the function of Na+/K(+)-transporting adenosine triphosphatase: an attempt at an integration into the Na+/K+ pump mechanism.

The particular aim of the review on some basic facets of the mechanism of Na+/K(+)-transporting ATPase (Na/K-ATPase) has been to integrate the experimental findings concerning the Na(+)- and K(+)-elicited protein conformation changes and transphosphorylations into the perspective of an allosterically regulated, phosphoryl energy transferring enzyme. This has led the authors to the following summarizing evaluations. 1. The currently dominating hypothesis on a link between protein conformation changes ('E1 in equilibrium with E2') and Na+/K+ transport (the 'Albers-Post scheme') has been constructed from a variety of partial reactions and elementary steps, which, however, do not all unequivocally support the hypothesis. 2. The Na(+)- and K(+)-elicited protein conformation changes are inducible by a variety of other ligands and modulatory factors and therefore cannot be accepted as evidence for their direct participation in effecting cation translocation. 3. There is no evidence that the 'E1 in equilibrium with E2' protein conformation changes are moving Na+ and K+ across the plasma membrane. 4. The allosterically caused ER in equilibrium with ET ('E1 in equilibrium with E2') conformer transitions and the associated cation 'occlusion' in equilibrium with 'de-occlusion' processes regulate the actual catalytic power of an enzyme ensemble. 5. A host of experimental variables determines the proportion of functionally competent ER enzyme conformers and incompetent ET conformers so that any enzyme population, even at the start of a reaction, consists of an unknown mixture of these conformers. These circumstances account for the occurrence of contradictory observations and apparent failures in their comparability. 6. The modelling of the mechanism of the Na/K-ATPase and Na+/K+ pump from the results of reductionistically designed experiments requires the careful consideration of the physiological boundary conditions. 7. Na+ and K+ ligandation of Na/K-ATPase controls the geometry and chemical reactivity of the catalytic centre in the cycle of E1 in equilibrium with E2 state conversions. This is possibly effected by hinge-bending, concerted motions of three adjacent, intracellularly exposed peptide sequences, which shape open and closed forms of the catalytic centre in lock-and-key responses. 8. The Na(+)-dependent enzyme phosphorylation with ATP and the K(+)-dependent hydrolysis of the phosphoenzyme formed are integral steps in the transport mechanism of Na/K-ATPase, but the translocations of Na+ and K+ do not occur via a phosphate-cation symport mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)